PRRSV美洲型经典株、变异株和疫苗株多重RT-PCR鉴别检测方法的建立及应用

Establishment and Application of a Multiplex RT-PCR Assay for Differential Detection of Classical,Highly Pathogenic and Vaccine Strains of North American Genotype PRRSV

为建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的快速鉴别检测方法,本研究针对PRRSV美洲型经典株、高致病性变异株以及TJM-F92疫苗株的Nsp2基因序列特点,设计2对特异性引物。经优化反应条件后,建立了能同时检测并区分PRRSV美洲型经典株、变异株及疫苗株的多重RT-PCR方法。该方法特异性强,与猪其他病毒间不存在交叉反应;敏感性高,对重组质粒标准品的检出下限为1.13×103拷贝/μL。应用所建立的方法对349份临床疑似病料进行检测,结果检出PRRSV阳性119份,其中美洲型经典株5份、变异株107份、TJM-F92疫苗株7份,且有变异株和TJM-F92疫苗株混合阳性7份。表明本研究成功建立了PRRSV美洲型经典株、变异株及TJM-F92疫苗株的多重RT-PCR鉴别检测方法,可用于PRRSV的临床检测及流行病学调查。

To establish a rapid method for differential detection of classical,highly pathogenic and TJM-F92 vaccine strains of North American genotype porcine reproductive and respiratory syndrome virus（PRRSV）,a multiple RT-PCR assay was established.In this assay,two pairs of primers were designed according to the genomic sequences of classical,highly pathogenic and TJM-F92 vaccine strains of PRRSV.The assay could only detect PRRSV,but not detect CSFV,FMDV,PRV and PCV2.The detection limit of the method was as little as 1.13×10^3 copies/μL of templates.The established assay was successfully used to detect 349 clinical samples and 119 samples were positive for PRRSV,of which 5 samples were positive for classical PRRSV（C-PRRSV）,107 samples for highly pathogenic PRRSV（HP-PRRSV）and 7samples for TJMF92 vaccine strain（V-PRRSV）,while 7samples were positive for HP-PRRSV and V-PRRSV.The results indicated that the established multiple RT-PCR assay could be used for differential detection and epidemiological investigation of PRRSV.